The Biocompatibility of Collagen-Glyco Semi-Interpenetrating Polymer Network Corneal Substitutes in Vitro and in Vivo

ObjectiveTo evaluate the biocompatibility of Collagen-Glyco Semi-Interpenetrating Polymer Network corneal substitutes in vitro and in vivo and explore its feasibility as corneal scaffolds.MethodsPart1Biocompatibility studies in vitro, selecting one of the white rabbits, male or female,2.0-2.5kg, provided by the Basic Experimental Animal Center of Tianjin Medical University, exclude corneal disorders. Collagen-Glyco Semi-Interpenetrating Polymer Network corneal substitutes was provided by the department of Materials Science and Engineering of Tianjin University. Material properties:diameter12mm, thickness is300μm, white light transmittance is more than90%, Water content is more than90%, tensile strength is0.6±0.1mPa, residual mass is over40%in30hours in vitro collagenase (12μg/ml) degradation test. Material:Sumianxin (xylidinothiazoline, ethylene diamine tetra acetic acid (EDTA), DHE hydrochloride and haloperidol compound) and ketamine was injected for intramuscular anesthesia. Limbal epithelium was cut and teared off using tunnel knife under a microscope, and then cut into the size of1mm x1mm, put uniformly in the semi-permeable membrane material. Corneal epithelial cell growth rate and morphology was observed by inverted microscope, cell line was stained by hematoxylin Seiichi eosin after two weeks.Part2Biocompatibility studies in vivo.48rabbits underwent anterior lamellar keratoplansty implanted the Collagen-Glyco Semi-Interpenetrating Polymer Network corneal substitutes in the experiment group,48rabbits underwent allogeneic anterior lamellar keratoplansty as the control group, the remaining24rabbits for the lamellar donor. We observed them after keratoplasty3d,7d,15d,1m,3m,6m by naked eye and slit lamp, observation inform include:corneal transparency and neovascularization after3d,7d,15d,1m,3m.6m. and then we killed eight animals at every time point in each group, rabbit corneal tissue were fixed in10%formaldehyde solution, stained by HE. Pathology and epithelial immunohistochemical of marker protein K3were observed using the light microscope.ResultsPart11. Inverted microscope observation, the tissue cells organized to single layer after five days and showed membranous growth, had obvious growth boundaries, the appearance like beach, cells interconnected, the area close to the organization was stratified growth, continued cultured for2weeks, the cells were stratified growth. The gas-liquid interface culture formed the continuous epithelial cell layer, connection between cells and carrier is solid.2. HE staining showed corneal epithelial structure of stratified squamous epitheliumPart21. Corneal transparency increased gradually after the operation between experimental and control group. It has significant statistically differences in corneal transparency between the two groups at3d,7d,15d postoperatively(P<0.05). After1month,3months and6months compared with the control group, it has no statistically difference at3d.7d,15d postoperatively (P>0.05).2. Corneal neovascularization score increased gradually at3d,7d,15d after the operation, reached the highest after1m, then decreased gradually in the experimental group. The score increased gradually in7d,15d1after operation and reached the highest, and then diminished gradually in the control group. It has significant statistically differences in corneal neovascularization score between the two groups at,7d,15d postoperatively(P<0.05). It has no statistically difference at other time postoperatively (P>0.05).3. Most corneal epithelium was defect in the experimental group after3d, peripheral epithelium grew to the central, uniform red stain of collagen covered the surface of the material, stroma edema, inflammatory cells is obvious in the graft incision. Monolayer epithelial cells covered the material7days after surgery, collagen fibers arranged in disorder, a large number of fibroblasts is in the stroma. After15d, there is still more stromal fibroblasts, collagen fibers arranged is still in disorder. lm after surgery, the corneal epithelium covered the superficial completely, stroma collagen fiber arrangement tends to rule, but still has abnormal matrix disorder.3m after surgery, epithelial repair, stromal collagen fibers arranged in rules.6m after surgery, collagen fiber arranged well and some areas still has small collagen disorders.3d after surgery, the corneal epithelium has some local defects in control group, stromal edema, and a slight increase in fibroblasts.7d after corneal epithelial healing, stromal edema, and the number of fibroblasts increased obviously.15d after surgery, epithelial becomed integrity, stroma collagen fibers arranged in the basic rules, the number of fibroblasts decreased.1m after surgery, the arrangement of collagen fibers is in consistent.3m and6m after surgery, the stroma collagen arranged in rules.4. immunohistochemistry showed that there is positive expression of the corneal epithelial cell-specific marker proteins K3after6months.ConclusionsCollagen-Glyco Semi-Interpenetrating Polymer Network corneal substitutes can support cornea epithelial growth, the connection between the cells are tight, the cells can form a continuous epithelium. The material can support the growth of the rabbit corneal epithelium and promote active remodeling of the corneal stroma,the next step we will improve the stability of the material in vivo, enhance its mechanical strength, and reduce the immunity of the material.So,Collagen-Glyco Semi-Interpenetrating Polymer Network corneal substitutes has good biocompatibility in vitro and in vivo, it is expected to become a new type of corneal scaffolds for further improvement.